The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0–3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia.
The biosynthesis of terpenoid natural products begins with a carbocation-based cyclization or prenylation reaction. While these reactions are mechanistically similar, there are several families of enzymes, namely, terpene synthases and prenyltransferases, that have evolved to specifically catalyze terpene cyclization or prenylation reactions. Here, we report that bacterial diterpene synthases, enzymes that are traditionally considered to be specific for cyclization, are capable of efficiently catalyzing both diterpene cyclization and the prenylation of small molecules. We investigated this unique dual reactivity of terpene synthases through a series of kinetic, biocatalytic, structural, and bioinformatics studies. Overall, this study unveils the ability of terpene synthases to catalyze C-, N-, O-, and S-prenylation on small molecules, proposes a substrate decoy mechanism for prenylation by terpene synthases, supports the physiological relevance of terpene synthasecatalyzed prenylation in vivo, and addresses questions regarding the evolution of the prenylation function and its potential role in natural product biosynthesis.
A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.
A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To define the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate cyclization patterns in bacterial type II TSs, along with detailed bioinformatics analysis of these TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.
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